Columns for SEC/GPC

[jabolko]

Hello to all,

i am not expert with SEC/GPC and I am not so familar with diferent columns for SEC. I would like to ask if anyone know or he using columns for separation of polymers which hydrodynamic volumen is less then 2000 Da. Can you recomend me one? Sample are soluble in THF, DMAc and MeOH.

I would be really gratefull for all answers.

bye

Jabolko

[unmgvar]

in general SEC columns with 5u can separate close too or baseline to baseline polymers whose sizes are of a ratio of 1:2
if you can find a 3u column then it is best
you need to get also the column that work best for the range that you want to use
for you either a 50 or 80A column should best

it is possible that you will need 2 or 3 columns it is not uncommon especially if you cannot find a 5u or less column
you will probably need a polymer based column. check that you can switch solvents has they tend to swell or shrink if not used with the correct solvent

[jabolko]

Thank you unmgvar,


I would like to ask you another question. So if I understand you good, if i find column with particle size 3u, I need more then two coulmns for good separation, is that correct?
So If I have two columns, they have to be same particle size? The same porosity? Two columns with same separation range? Can you please explain me this. What is inportant when we put two or more columns together?

Thank you again for all answers.

jabolko

[unmgvar]

all depends on your sample
if you have a very low ploydispersity on you compounds and the Mw ratio between them is 1:2 or higher then you will get with a 5u or less particle size column a good separation. and 1 column will be sufficient
but if you have a high polydispersity or a less then 1:2 Mw ratio then you will need more then one column.
depending on the sample it will be 2-3 columns and we still we could be without baseline separation
all depends on the compounds characteristics

[jabolko]

Hello,
so if i have Mw/Mn around 1,04 to 1,7 is one column enought, but I have that one with particle size 5u, do you think with 3u it be better? What about if use for separation two columns first I take column with particle size 5u and second one with 3u??? Any oppinion about that??
And in another case Mw/Mn is 5, 7 so I need more columns. Sould be that ok if put one colum in seperation range from 100-30 000 Da (1) and another one in range 100-100 000Da (2)? In order that I put first columne (1) and then (2)? Or it have to be other way around?
Does anyone know if maybe linear columns be better for separation for polymers with hydrodynamic volumen less then 2000 Da?

Thanks agin for, I am really gratefull for all answers. Thank you all.

jabolko

[unmgvar]

jabolko here is a link to a protein application that can show you how SEC behaves
the peaks here are made of very clean very low dispersity compounds
this is a protein application with a column suited for buffers
http://www.sepax-tech.com/application_notes/ZE1008.pdf

[jabolko]

Thank you unmgvar for all answers and for link.

[gstaepels]

If you are running THF, I would recommend PL-Gel mixed bed type E columns. They are available through Agilent. I use these column for years in a set of 3 and achieved very good results. I would not recommend these using DMAc at 85°C. My experience with these columns in THF is they they last for several years.

[jabolko]

Dear Gilbert,

thank you for you help. So i have to buy two columns PLgelMIXED-E 3um and i will get good separation for polymers which hydrodynamic volumen si less then 2000 Da?

Thanks

Jabolko

[jabolko]

Dear Gilbert,
one more question. Is better to buy 7,5 mm ID or 4,6 mm ID? is that depends only how much mobile phase you use in each case or have other influent?
thanks

jabolko

[gstaepels]

Dear Jabolko,

I use 7.5 mm ID columns. I have tried 4.6 mm ID columns, but I was not satisfied. I think that the separation in GPC is the ratio between the column volume outside the particle and the volume of the column and the pores.
If you are concerned about solvent consumption, I use a Waters RI detector, which has a switching valve to direct the eluent flow to the solvent reservoir or to the waste can. This reduces solvent consumption.

[jabolko]

Dear Gilbert,
thank you, i am going to listen yours recommendations, you have experiance I am working with SEC/GPC only for a half year . And that polymers are not so easy to determ becouse they are small.
Thank you, i am really gratefull for all tips you gave me.
Bye

jabolko

[jabolko]

Dear gstaepels,

I order then column PLgelMIXED-E 3um (thanks for recomending it), I read specifications and I think it will solve probleme and I get ok separation. But here I have another probleme . I read that every THF isnt proper for storage that column, can you explain me something about this? I have that THF (TETRAHYDROFURAN CHROMASOLV (>99,9%)SL-34865-2,5L,SIGMA-ALDRICH).

Many thanks for all answers.

jabolko

[gstaepels]

Jabolko,

When I am running "standard GPC" (= in THF with RI detector), I use THF AR from Merck. This THF contains 250 ppm BHT. I run the columns at 40°C. When I need to use the ELSD, I use THF ULC/MS from Biosolve. I have found that this grade of THF gives me the smoothest baseline, comapred to THF from other vendors (Merck, Prolabo, Aldrich). Also the price/liter of the Biosolve THF (in bottles) is much lower (The Merch THF AR is bought in 25 liter drums).
These are my experiences and they are not necessarly a scientific facts.

[jabolko]

Dear Gilbert,

so you use for standar GPC THF AR from Merck in which you do sample analyse and then in the end you storage column in that THF? So Biosolve THF dont contains 250 ppm BHT?

I know when you read that questions you will think taht they are very silly but I would be really gratefull if you tell me how you do it taht i can see if i am doing right.

They say is stupid who dont ask. So I would like to ask you next questions:
-Do you do calibration curve every time you measuring samples, or for same method and column you dont do it every time?
-For that column PLgelMIXED-E 3um or other columns (How do you prepare standards? What kind of weight you use for standards and samples. What is initiation concentration of samples? Do filtrate sample thru 0,45um PTFE? )

Thanks for all your answers and help and thanks in advance for all answers.


Jabolko